Cholera toxin (CT), produced by Vibrio cholerae, induces the life-threatening diarrhea of cholera. CT travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic A1 subunit then dissociates from the rest of the toxin, unfolds, and passes through an ER translocon pore to reach its cytosolic Gsa target. Translocation into the cytosol is facilitated y the quality control system of ER-associated degradation (ERAD). Most ERAD substrates are extracted from the ER through a mechanism involving the cytosolic AAA ATPase p97. However, p97 appears to play a minimal role in CTA1 translocation. The overall goal of this project is to define the molecular mechanism for CTA1 translocation and its subsequent activation in the cytosol. We recently reported that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. This work established a new role for Hsp90 in the extraction of a soluble ERAD substrate from the ER. Hsp90 works with Hop and Hsc70 to refold client proteins. Based upon our published and preliminary data, we hypothesize Hsp90, Hop, and Hsc70 form a core translocase complex that directly facilitates CTA1 translocation to the cytosol. We further predict the Hsp90/Hsc70-assisted refolding of disordered proteins is linked to their translocase function: by coupling translocation with refolding, Hsp90 and Hsc70 would prevent the (re)folded CTA1 protein from sliding back into the translocon pore. This process would provide the driving force for CTA1 translocation. We also predict the Hsp90/Hsc70-assisted refolding of CTA1 will place the cytosolic toxin in a conformation that can be activated by host ADP-ribosylation factors (ARFs). Finally, we predict the Hsp90/Hop/Hsc70 complex is also involved with the ER-to-cytosol export of other toxins and endogenous ERAD substrates that utilize a p97-independent translocation pathway. In this application, we will (i) define the core components of the translocase complex and their binding sites on CTA1; (ii) demonstrate the refolding function of the translocase complex and examine its potential effect on ARF-stimulated toxin activity; and (iii) identify a broader range of toxins and endogenous ERAD substrates for the translocase complex. Our structure / function analysis of the translocase complex will employ a unique combination of molecular microbiology, cell biology, and biophysics. This project will provide molecular insight into the poorly understood process of CTA1 translocation and will define a new route for the ER-to-cytosol export of ERAD substrates.